Original karst tiankeng with underground virgin forest as an inaccessible refugia originated from a degraded surface flora in Yunnan, China

Karst tiankengs are rare natural habitats, having a local microclimate different from surrounding regions. A contrast study on plant communities at the inside and outside of the primitive tiankeng was carried out by performing the qualitative analysis of species compositions of arborous and shrub layers. We found that plant communities in the primitive tiankeng belong to the subtropical moist evergreen broad-leaf forest, whereas those outside the tiankeng belong to subtropical semi-moist needle-broadleaved mixed forest. Trapped habitat of primitive karst tiankeng protects the plant communities significantly different from those in external karst ecosystems, so karst tiankeng has the great value in the plant species protection. Although the trapped habitat decreases plant species abundance inside tiankeng to some extent, highly diverse shrub species are present in the inside-tiankeng plant communities, and the primitive karst tiankeng plays an important role in the plant diversity protection. The primitive karst tiankeng is an important refugia for plant not only as a plant species protection library, but a plant diversity protection library. When implementing measures for the reduction of damages to biodiversity due to global climate changes and human activities, more attention should be paid to the primitive karst tiankeng as a small ecological refugia and biodiversity protection library.

Plant composition outside the primitive tiankeng. 8 arbor species (e.g., Cunninghamia lanceolata, Pinus yunnanensis and Quercus semecarpifolia) that belong to 7 genus (e.g., Cunninghamia, Pinus and Juniperus) and 5 families (e.g., Taxodiaceae, Pinaceae and Cupressaceae), and 25 shrub species (e.g., Myrsine africana, Cotoneaster franchetii and Viburnum propinquum) that belong to 18 genus (e.g., Myrsine, Podocarpium and Jasminum) and 13 families (e.g., Myrsinaceae, Leguminosae and Oleaceae) were identified (Table 3).   For arbor species, non-overlapping significant differences were observed in "Family" between inside (5 families) and outside (12 families) the tiankeng (Fig. 1). For shrub species, 14 families inside and 13 families outside, including 3 sharing families (Leguminosae, Cornaceae, and Berberidaceae) were determined (Fig. 2). Among the 3 sharing families, only existed one shared genus (Dalbergia). In other words, significant plant composition differences existed inside and outside the primi-   Plant diversity inside and outside the primitive tiankeng. α-Diversity index of plant communities inside the tiankeng. Plant diversity inside the tiankeng was investigated by sampling the entire site. Data collections were performed by the adjacent gridding method. The α-diversity index of each sample plot was calculated, and the mean was used as the α-diversity of the entire plant communities at the bottom. Based on the means of α-diversity index, the test results of Margalef abundance, Shannon-Wiener diversity, Pielou evenness, and Simpson dominance in the arborous and shrub layers at the bottom were generally consistent (Table 4). Statistical differences ranged from 23.3 to 34.4% in the arborous layer, and 30.4 to 52.1% in the shrub layer. For both arborous and shrub layers, obvious differences of abundance, diversity, and species composition were observed  α-Diversity index of plant communities outside the tiankeng. Four sample plots were set outside the primitive "Damaosi" tiankeng to investigate plant diversity. Margalef abundance, Shannon-Wiener diversity, Pielou evenness, and Simpson dominance of the arborous layer in southwestern, northwestern, southeastern, and northeastern parts had minimal differences (Fig. 4). The variable coefficients of the indexes in all directions only ranged from 5.0 to 14.7%, indicating that the arborous layer is relatively uniform with respect to species abundance, diversity, and species composition in a trapped topography. Shannon-Wiener diversity, Pielou evenness, and Simpson dominance of the shrub layer on four directions were relatively uniform, the variable coefficient ranged from 2.5 to 19.1%. However, Margalef abundance varied dramatically, and the variable coefficient reached as high as 30.6%. The extent of variation on the value of Margalef abundance was in the following order: southeast > northeast > southwest > northwest. In other words, the plant abundance of the shrub layer in the eastern part outside the tiankeng was higher than that in the western part, and the plant abundance of the shrub layer in the southern part was higher than that in the northern part.
Based on comparison of α-diversity index between the arborous and shrub layers outside the tiankeng, the shrub layer showed higher plant abundance (S = 2.246 > A = 1.218), and the arborous layer showed higher species diversity (S = 0.523 < A = 1.639) ( Table 5).
Comparison between the α-diversity indexes obtained inside and outside the tiankeng. With respect to arborous species, the Margalef abundance, Shannon-Wiener diversity, Pielou evenness, and Simpson dominance outside the tiankeng were all higher than inside. A great gap between the areas outside and inside the tiankeng with respect to species abundance was observed. These results reflect that the arborous species diversity in areas outside the tiankeng is higher than that inside. For shrub species, Shannon-Wiener diversity and Pielou evenness were higher in areas inside than those outside, whereas Margalef abundance and Simpson dominance showed the opposite result. In summary, higher arborous species diversity was observed outside the primitive "Damaosi" tiankeng, but higher shrub species diversity was observed inside it (Fig. 5).

Discussion
The theory of island biogeography proposed by MacArthur and Wilson in 1967 is one of the most important theories in ecological study 35 , and provides important theoretical references for biodiversity protection. It was initially employed to study biodiversity in ocean islands, then gradually used to study biodiversity in "Continental islands", such as high mountains, natural conservation areas, lakes, and forests with surrounding open areas 36,37 . Naturally occurring habitat fragmentation in the karst region is prominent 38 . Raschmanová 34 and his research team showed that, in karst geomorphological landscapes, dark, wet, and cold habitats in a doline are similar to a typical island habitat in temperate areas found in the middle of Europe. The special internal microclimate in karst tiankeng with good trapping and high vertical depth showed obvious "island" characteristics with surrounding regions. Culver studied karst environment and found that the vertical topography in karst landscape can intercept extreme climate events to some extent and can lower extreme temperature significantly in the local area. This conclusion strongly supports the "island" characteristic of karst tiankeng 39 . Moreover, such "island" habitat provides an important place for survival and reproduction of animals and plants, and especially can become the ecological refugia of cold-resistant plants under climate change [27][28][29][30][31][32] . Daily showed that the changing land utilization pattern causes landscape fragmentation and ecosystem simplification and separates biotic populations from communities, resulting in the sharp reduction of global animal and plant diversity 40 . Although the topography in tiankeng causes local habitat fragmentation, the vertical steep precipices and trapped morphology fail to make the primitive "Damaosi" tiankeng an isolated "island" with poor ecological environment. On the contrary, the primitive "Damaosi" tiankeng possesses abundant plant species and the vegetation coverage reaches about over 95%. Arbor and shrub species inside and outside differ significantly. The vegetation types in the area of tiankeng group are mainly dominated by subtropical semimoist needle-broadleaved mixed forest, whereas the dominant vegetation types inside the primitive "Damaosi" tiankeng are moist evergreen broad-leaved ones in the subtropical evergreen broad-leaved forest, which mainly occur in moist monsoon climate regions and its development would be restricted in the middle and east plateaus of Yunnan 24 . However, protected by primitive "Damaosi" karst tiankeng's natural precipices, the native vegetation types were maintained, and the plant communities are resistant to the effects of climate change and human activities. Bartgis 41 discovered some plant species that are rare or endangered in local areas could be found in sinkhole pond. Yu 42 and her team studied Xylophyta diversity in the ecological niche in the Stone Forest Geopark in Yunnan Province, and found that ecological niches like crack ditches, deep karst pit, sphenoid trough, and corrosion clitter still maintain some local plant propagation after suffering from strong human disturbances, whereas local plant propagation of other ecological niches are absent. Dang 43 studied the origins and evolution of flora in 14 tiankengs in the Guangxi Dashiwei tiankeng group and discovered 1 new genus and 5 new species. Our results on plant communities in the primitive tiankeng also proved that a karst tiankeng formed by longtime tectonic motions is not only a kind of unique and large topography but also a precious species protection library. It is an important material to study vegetation evolution in tiankeng areas and an important plant species protection library.
Climate change and habitat loss cause serious threats to global biodiversity and ecosystem. Protecting habitat integrity and species refugia is crucial to wild animals and plants. Climate change has caused the migration of plants on biotic formation. Thus, more attention must be paid not only to existing large refugia but also to small refugia in some landscape areas and small natural conservation areas. Tiankeng is one of rare natural habitats, and its unique habitats are precious refugia for plant species and diversity 44 . This species sanctuary function has www.nature.com/scientificreports/ important value on cope with future climate changes and vulnerable ecological environment in karst regions, thus karst tiankengs are important protected objects of small ecological refugia. Diversity of microenvironment in one community environment is positively correlated with community abundance. Compared with outside-tiankeng habitat, inside-tiankeng habitat are limited and local environmental heterogeneity is weak. Thus, the inside-tiankeng microenvironmental diversity is lower than outside-tiankeng. Moreover, due to the attributes of depth and vertical trapped precipice, internal illumination, temperature, wind speed, and other environmental resources are inferior in the inside, resulting in low species abundance inside the tiankeng. Meanwhile, the "intermediate disturbance hypothesis" suggests that certain disturbances from human beings or nature contribute to species diversity growth. Connell and Lawton 45 disclosed that species abundance is related with disturbances to the crown canopy. Species abundance decreases upon strong interferences to forest stand, and forest community is unable to reach the mature forest form. Possibly, few species are present in slightly disturbed forests, but the species abundance of forest increases to some extent after moderate disturbance. Helmus 46 and his team took the chameleon population on the Caribbean Islands as the example and found that biodiversity on islands is sensitive to human disturbance except for separation degree and island areas. The immigration of external species due to human activities increases biodiversity on islands, and thus human activities considerably affect biodiversity and abundance in habitats in isolated islands. "Damaosi" tiankeng is primitive, and its internal ecosystem has been slightly disturbed by human activities. And the tiankeng group is in the Haifeng Natural Conservation Area where human activity is limited, so the outside-tiankeng plant community suffer slight or intermediate disturbances. As a result, the low species abundance inside the tiankeng might conform to the "intermediate disturbance hypothesis. " According to comparison of species abundance outside the primitive "Damaosi" tiankeng on different directions, the shrub abundance in the east is higher than that in the west, and the shrub abundance in the south is higher than that in the north. The results of the field investigation showed that the primitive "Damaosi" tiankeng is located on the inclined concave slope surface of groove head of the small river basin and marginal terrains and are characteristic of "high in the north and low in the south". And the "Damaosi" tiankeng has different soil corrosion characteristics. The greatest number of flat terrains and highest frequency of soil accumulation are observed in the southeast. Slope is the main terrain factor that influences the surface distribution of vegetation 47 , such as grass, vine, and shrubs. Moreover, soil accumulation is the key condition for vegetation growth and plant community succession in the south karst region 48,49 . Fewer limestone, more soil accumulations, and better water retention capacity are present in the east part of outside tiankeng compared to west. Thus, the former is more favorable for plant growth and has higher species abundance. Du 50 discussed the coupling relationship between plant diversity and soil properties in a karst vulnerable ecosystem, and they found that soil minerals and microorganisms both regulate vegetation composition, community type, and growth conditions. Conversely, soil properties also can change with vegetation. Therefore, vegetation plays an important role in ecological recovery, conservation of water and soil, and soil accumulation in karst regions with serious stony desertification problems in China.
According to our results, arbor species diversity is higher outside the primitive "Damaosi" tiankeng, but shrub species diversity is higher inside. Illumination is an essential factor for plant growth and photosynthesis. The vertical precipices and trapped topography in karst tiankeng block illumination, and shrub species possess higher survival advantages in such "underground forest" than arborous species. Studies on plant diversity of different ecosystems in karst fengcong depression disclosed that maximum species diversity occurs in secondary forests. The primitive "Damaosi" tiankeng has a primary forest inside it and a secondary forest outside. Although trapped habitat conditions decrease plant abundance to some extent, shrub species diversity inside the tiankeng is still higher than that in the outside, thus, the unique habitat of karst tiankeng is an important plant diversity protection library. Dolinar 33 studied the flora of vascular plants in some phytogeographic regions in Solvenian, and concluded that the abundant plant diversity in this region is mainly attributed to the protection of karst ponors. Shui 8 and his team discovered that plant diversity in the habitats of karst tiankeng is different from common karst topographies such as dolines and sinkholes. Liu 51 and his team also pointed out that tiankeng has higher plant abundance than other karst depression. Many studies reported that karst dolines are important species diversity protection libraries 27,28,33 . A karst tiankeng, which has larger size and higher closure and higher isolation degree with its surrounding areas, is believed to possess an exceptional value in species diversity protection. The karst region in south China is not only an important basis for biodiversity protection but also an important habitat for rare and endangered animals and plants. It possesses significant world heritage value to protect biodiversity. Meanwhile, the role of karst tiankeng as a small biodiversity protection library should be emphasized further.

Methods
Study area. The Zhanyi tiankeng group lies in the Dapo Town within the Haifeng Natural Preservation Area in the Yunnan Province. This area belongs to the Jinsha river system and control area of the Niulan River Basin, which is the first-level tributary of Jinsha river (Fig. 6). It has a typical subtropical plateau monsoon climate, having windy weather and drought in winter and spring; wet, warm, and rainy weather conditions in summer and autumn; and small annual temperature differences and large daily temperature differences 52 . This climate lays an important basis for the formation of abundant plant diversity.
On August 2016, the research team made a field investigation in Zhanyi tiankeng group in Yunnan Province. Ten tiankengs were investigated, and the basic data of some tiankengs were collected (Table 6). Tiankeng I ("Damaosi") is a primitive tiankeng that has vertical precipices and get few human disturbances. According to official measurement, its maximum diameter can reach approximately 200 m, and in this survey, the longest diameter measured was 136.8 m. Tiankeng II and Tiankeng III which have slightly disturbed by human activities, and is called "middle tiankeng" and "small tiankeng, " respectively. Tiankengs IV, V, and VI are moderately degraded tiankengs and are called by locals as "Huoshipo, " "Bajiaxiantang, " and "Shenxiantang, " respectively.  www.nature.com/scientificreports/ Previously, the bottom of these degraded tiankengs had been used for agriculture. Currently, the farmlands reverted to woodlands, and vegetation recovered to some extent. Tiankeng VII is a seriously degraded tiankeng and is called by locals as "Dazhujing", it is large and have a maximum diameter of about 455 m. Its bottom contains one blind river and several karst caves. Tiankeng VIII is a degraded doline and is in the west of "Bajiaxiantang". In this paper, the primitive "Damaosi" tiankeng, which has hardly influenced by human activities, was chosen as the research object to study the tiankeng's plant diversity (Fig. 7).
Sample design. 30 m × 30 m sample plot was established for the census of community at the outside-tiankeng habitat, and these quadrats was distributed in the southwestern, northwestern, southeastern, and northeastern parts of the primitive tiankeng, respectively (Fig. 8). In each sample plot, five 10 m × 10 m shrub sample sites were set in a "pentalobe" pattern. The arborous layer was studied by a full sample survey (Fig. 9). The bottom area of "Damaosi" tiankeng was approximately 0.8 × 10 4 m 2 . Plant species at the bottom were investigated. For the convenience of data statistics, 17 sample sites (20 m × 20 m) were set by the adjacent gridding method, and plant species in each site were investigated. Given the poor accessibility of a primitive tiankeng, an unmanned aerial vehicle and high-resolution photographs technique were used to collect data from the bottom for plant species survey. We then mainly focused and analyzed on the arborous and shrub layers.

Identification of species.
With field sample plot setting, species survey and data statistics, we performed the preliminary field species identification, and unidentified species were identified in the laboratory on the basis of Flora of China, Yunnan Local Flora, "China Herbarium" "China Plant Species Database" and "PPBC China Plant Image Library". Those remained unidentified were subjected to further consultations from experts for three times. Two consultations were performed online, one of which was performed by holding an expert consultation conference. In this conference, unidentified plant species were presented to experts in phytological and ecological fields. After the conference, the opinions of the experts were evaluated and summarized to obtain useful data. Consultants who are expert in plant species identification were mainly selected by two ways. First, experts in related fields were consulted. Second, experts were assessed by reading his articles. A total of 22 expert testimonies were sent, and 15 of which were collected effectively. Plant species identification received strong support from 10 senior experts and 5 scholars with PhD, all of them have the outstanding achievements in bioscience and ecology fields.
Data analysis. Evolutionary tree construction. The Latin name lists of sample species were entered into the Phylomatic platform, and constructing the species evolutionary trees by its database, which is based on the APG III, it can automatically constructing the phylogenetic topology of species, integrating the evolutionary tree information according to the slik algorithm, and outputting the evolutionary trees with branch lengths. It is an index that reflects species abundance in a biocommunity (sample).
where D is the Margelef richness index; S is the number of species; N is the total number of individuals.